A bootstrapping resampling technique was employed to demonstrate the variability in sampling distribution of mean type I and II FCSAs. Soleus and extensor digitorum longus (EDL) muscle cross-sections were stained for myofibrillar ATPase activity to identify fiber type (either type I or II ) and laminin to facilitate FCSA measurements. These studies were conducted in twenty-three, male Fisher 344 x Brown Norway F1 hybrid (F344BN) rats. In this report, we first identified the variability in mean estimates of FCSA as a function of the number of fibers measured on hindlimb muscle cross-sections, and secondly evaluated whether counting a subset of fibers within cross-sections could predict total cross-sectional FN. The objective of this study was to inform decisions about how many fibers to measure in order to obtain a reliable estimate of FCSA and FN. However, the reliability of estimating FCSA and FN from only subsets of fibers in rat hindlimb muscle cross-sections has not been systematically evaluated. Notably, this representative subset can vary from as few as 25 to as many as hundreds of fibers. What many research groups have done is to estimate FCSA and/or FN based on a representative subset of single muscle fibers within a muscle cross-section. Thus, as these procedures are time-consuming and labor-intensive, they can result in higher costs to conduct the research. This work also involves the careful identification and evasion of areas distorted by freezing, sectioning, processing, or staining which may affect the accuracy of FCSA and FN measurements. These measurements are performed by experienced operators visually analyzing successive histological muscle cross-sections which contain hundreds to thousands of single muscle fibers. The study of skeletal muscle atrophy and hypertrophy in the laboratory rat commonly involves 1) the assessment of single muscle fiber size, referred to as the cross-sectional area of skeletal muscle fibers (FCSA), and 2) the number of single muscle fibers in a muscle cross-section (FN). Counting 15-20% of the fibers in cross-sections provides a reasonably reliable estimate of the total fiber number. These data describe a pattern of improved precision in estimating mean fiber cross-sectional area as sample size of fibers measured increases to at least 150 in this rat model. Fiber numbers from 3 to 4 fields (approximating 15 to 20% of the cross-section) provided a reasonably predictive value of total fiber number ( r=0.57-0.59, P=0.003). In the same muscle sections, total fiber number was compared to fiber numbers within 1, 2, 3, and 4 fixed field areas (10x magnification 1000 x 1500 pixels in size/field) on the cross-section. Coefficients of variation for bootstrapped mean type I fiber cross-sectional areas were lower than for type II. A bootstrapping resampling technique demonstrated that variability in sampling distribution of mean type I and II fiber cross-sectional areas decreased and gradually stabilized as the number of fibers measured increased with large declines in variability occurring at numbers below 150 fibers. Mean type II fiber cross-sectional area was based on EDL muscle sections which were predominantly composed of type II muscle fibers. Mean type I fiber cross-sectional area was based on soleus muscle sections which were predominantly composed of type I muscle fibers. We outlined the circumference of 1000 to 1600 single muscle fibers for measurement of fiber cross-sectional area within muscle sections. Soleus and extensor digitorum longus (EDL) muscle cross-sections from 23-month-old, male Fisher 344 x Brown Norway rats were stained for myofibrillar ATPase activity to identify muscle fiber type (either type I or II ) and laminin to facilitate fiber cross-sectional measurements. This study examined the variability in mean estimates of fiber cross-sectional area as a function of the number of fibers measured, and tested whether counting a subset of fibers in a cross-section could predict total fiber number in middle-aged rats. The reliability of estimating muscle fiber cross-sectional area (measure of muscle fiber size) and fiber number from only a subset of fibers in rat hindlimb muscle cross-sections has not been systematically evaluated.
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